Polyphenols are a highly diverse group of compounds (Ferreira, D., Steynberg, J. P., Roux, D. G. and Brandt, E. V., Tetrahedron, 48 (10), 1743-1803 (1992)) which widely occur in a variety of plants, some of which enter into the food chain. In some cases they represent an important class of compounds for the human diet. Although some of the polyphenols are considered to be non-nutritive, interest in these compounds has increased because of their possible beneficial effects on health.
For instance, quercetin has been shown to possess anticarcinogenic activity in experimental animal studies (Decshner, E. E., Ruperto, J., Wong, G. and Newmark, H. L., Carcinogenesis, 7, 1193-1196 (1991) and Kato, R., Nakadate, T., Yamamoto, S. and Sugimura, T., Carcinogenesis, 4, 1301-1305 (1983)). (+)-Catechin and (−)-epicatechin have been shown to inhibit leukemia virus reverse transcriptase activity (Chu, S.-C., Hsieh, Y.-S. and Lim, J.-Y., J. of Natural Products, 55 (2), 179-183, (1992)). Nobatanin (an oligomeric hydrolyzable tannin) has also been shown to possess anti-tumor activity (Okuda, T., Yoshida, T., and Hatano, T., Molecular Structures and Pharmacological Activities of Polyphenols—Oligomeric Hydrolyzable Tannins and Others—Presented at the XVIth International Conference of the Group Polyphenols, Lisbon, Portugal, Jul. 13-16, 1992). Statistical reports have also shown that stomach cancer mortality is significantly lower in the tea-producing districts of Japan. Epigallocatechin gallate has been reported to be the pharmacologically active material in green tea that inhibits mouse skin tumors (Okuda et al., Ibid.). Ellagic acid has also been shown to possess anticarcinogen activity in various animal tumor models (Boukharta, M., Jalbert, G. and Castonguay, A., Efficacy of Ellagitannins and Ellagic Acid as Cancer Chemopreventic Agents—Presented at the XVIth International Conference of the Group Polyphenols, Lisbon, Portugal, Jul. 13-16, 1992). Proanthocyanidin oligomers have been patented by the Kikkoman Corporation for use as antimutagens. The use of phenolic compounds in foods and their modulation of tumor development in experimental animal models has been recently presented at the 202nd National Meeting of the American Chemical Society (Phenolic Compounds in Foods and Their Effects on Health I, Analysis, Occurrence & Chemistry, Ho, C.-T., Lee, C. Y. and Huang, M.-T. editors, ACS Symposium Series 506, American Chemical Society, Washington, D.C. (1992); Phenolic Compounds in Foods and Their Effects on Health II. Antioxidants & Cancer Prevention, Huang, M.—T., Ho, C.-T. and Lee, C. Y. editors, ACS Symposium Series 506, American Chemical Society, Washington, D.C. (1992)).
However, these citations do not relate to cocoa extracts or compounds therefrom or to any methods for preparing such extracts or compounds therefrom, or to any of the uses described in U.S. Pat. No. 5,554,645 issued Sep. 10, 1996 to Romanczyk et al., U.S. Pat. No. 5,712,305 issued Jan. 27, 1998 to Romanczyk et al., and U.S. Pat. No. 5,650,432 issued Jul. 22, 1997 to Walker et al.
Isolation, separation, purification, and identification methods have been established for the recovery of a range of procyanidin oligomers for comparative in vitro and in vivo assessment of biological activities. For instance, anti-cancer activity is elicited by pentameric through decameric procyanidins, but not by monomers through tetrameric compounds. Currently, gram quantities of pure (>95%) pentamer are obtained by time-consuming methods. These methods are not satisfactory for obtaining sufficient quantities of the pentamer for large scale pharmacological and bioavailability studies. Even greater effort is required to obtain multi-gram quantities of higher oligomers (hexamers through decamers) for similar studies since their concentration in the natural product is much less than the pentamer. Additionally, increasing oligomeric size increases structural complexity. Factors such as the chirality of the monomeric units comprising the oligomer at different interflavan linkage sites, dynamic rotational isomerization of the interflavan bonds, conformational states of the pyran ring, and the multiple points of bonding at nucleophilic centers pose efficiency constraints on current analytical methods of separation and purification for subsequent identification.
For instance, previous attempts to couple monomeric units in free phenolic form using mineral acid as the catalyst in aqueous media have met with limited success. The yields were low, the reactions proceeded with poor selectivity, and the oligomers were difficult to isolate. (Stynberg, P. J., Nel, R. J., and Ferreira, D., Tetrahedron, 54, 8153-8158 (1998); Botha, J. J., Young, D. A., Ferreira, F., and Roux, D. J. J., J. Chem. Soc., Perkins Trans. 1, 1213-1219 (1981)).
Benzylated monomers have been prepared by methods described by Kawamoto, H., Nakatsubo, F. and Murkami K., Mokuzai Gakkashi, 37, 741-747 (1991) where benzyl bromide was used in combination with potassium carbonate (K2CO3), and dimethyl formamide (DMF). The yield, however, was only about 40%. In addition, competing C-benzylation leads to a mixture of products which makes isolation of the target monomer more difficult. Also, partial racemization of (+)-catechin at both the C-2 and C-3 positions was observed (Pierre, M.-C. et al., Tetrahedron Letters, 38: 32, 5639-5642 (1997)).
Two primary methods for oxidative functionalization are taught in the literature (Betts, M. J., Brown, B. R. and Shaw, M. R., J. Chem. Soc., C. 1178 (1969); Steenkamp, J. A., Ferreira, D. and Roux, D. J., Tetrahedron Lett., 26, 3045-3048 (1985)). In the older method, protected (+)-catechin was treated with lead tetraacetate (LTA) in benzene to produce the 4β-acetoxy derivative which was then successfully hydrolyzed to the 3,4-diol. Flavan-3,4-diols are incipient electrophiles in the biomimmetic synthesis of procyanidins. However, flavan 3,4-diols which have an oxygen functionality at the C-5 position are not available from natural sources and have to be synthesized. Oxidative functionalization of the prochiral benzylic position to form the 3,4-diols thus offers considerable potential in the synthesis of procyanidins. The major drawback of this reaction was a low yield (30-36%) of the acetate during the LTA oxidation. The more recent method of oxidatively functionalizing the C-4 position relies on the use of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). In this method, the protected monomer was treated with DDQ in methanol. This allows introduction of a methoxy group at the C-4 position in a stereospecific manner. The yield is about 40-50%.
There are a number of reports on the coupling reaction between monomers and their 3,4-diols in aqueous acid. These methods are unsatisfactory because of low yields, lack of specificity, and difficulty in the purification from aqueous media. Kawamoto, H., Nakatsubo, F. and Murakami, K., J. of Wood Chem. Tech., 9, 35-52 (1989) report the titanium tetrachloride (TiCl4) mediated coupling between 4-hydroxyl tetra-O-benzyl (+)-catechin and 5 equivalents (eq) of tetra-O-benzyl (+)-catechin to produce a 3:2 mixture of 4α→8 and 4β→8 dimers.
Hence, there is a need for synthesis methods which provide large quantities of structurally defined oligomers for in vitro and in vivo assessment. Such synthesis methods can lead to the creation of multiple configurational oligomers, some identical to those found in nature, as well as rare or “unnatural” types. Accordingly, it would be advantageous to develop a versatile synthetic process capable of providing large quantities of any desired procyanidin oligomer.